﻿STUDIES IN PLAGUE IMMUNITY. 241 



solution has been used. The method employed in making these suspen- 

 sions has been as follows: 



One cubic centimeter of an 0.85 per cent saline solution has been placed in a 

 small test tube and 1 oese of a 24-or 48-hour agar culture of the organism has 

 been introduced into the tube, thoroughly rubbed up against the walls by means 

 of the oese and the bacteria gradually moistened with the saline solution. While 

 the suspensions obtained with some pest cultures in this manner are satisfactory, 

 a complete suspension of the organism can not be produced with others and it 

 is necessary to combine the suspensions and either filter or allow them to stand 

 until the larger clumps of bacteria have settled to the bottom of the tube, when 

 the supernatant fluid may be drawn off with a pipette. 



However, while satisfactory suspensions are apparently obtained by 

 these methods, the greatest difficulty is experienced with certain cultures 

 of this organism in keeping the bacteria in suspension for a longer 

 period than, at most, one or two hours. After this time the organisms 

 begin to precipitate and sometimes at the end of two or three hours 

 the bacteria may have settled almost entirely to the bottom of the tube, 

 the supernatant fluid above appearing almost clear. This phenomenon 

 is particularly marked with the much attenuated plague cultures. 



I have followed the suggestion of Shibayama 65 of growing the bacillus 

 in the ice box at a temperature of from 5° to 8° C, with the object 

 of obtaining a less sticky and viscid growth of the pest bacillus and 

 thus securing a more satisfactory suspension of the organism in saline 

 solution. However, while I have found that with cultures which have 

 grown for a number of generations at such a tenrperature a much less 

 viscid growth apparently is obtained and one which may easily be 

 suspended in the saline solution, on the other hand the bacteria so 

 grown become spontaneously precipitated from the saline suspensions 

 in a much shorter time than do the organisms of the same strain grown 

 at a temperature of 30° C. I have also found that the addition of 

 normal serum to suspensions of the pest bacilli in saline solution some- 

 times retards and sometimes increases the tendency of the bacteria to 

 settle out of the fluid. For these reasons pseudo-reactions are not 

 infrequently encountered in performing agglutination tests with the 

 plague bacillus, and great care is sometimes necessary to distinguish such 

 reactions from those of true agglutination. Therefore, it is advisable 

 to perform in duplicate all tests for agglutination with the different 

 dilutions of the serum and at the same time to carry on a parallel series 

 of experiments in the same dilutions with a normal serum from an 

 animal of the same species and, moreover, to repeat all the tests with 

 another transplant of the same culture upon the following day. Only 

 by taking these precautions is it possible to distinguish certain pseudo- 

 agglutinations of the pest bacillus from true ones. When testing the 



°*Oentrll. f. Bakteriol. Orig. (1005), 38, 482. 



